The goal of this project is to define the relationship of the basic helix-loop-helix transcription factors Hand2 and Twist1 during mammalian lower jaw development. Our hypothesis is that Hand2 and Twist1 together establish an organizing domain within the mandibular pharyngeal arch that is required to drive lower jaw development. Within this domain, Hand2 and Twist1 act through effectors to accomplish a lower jaw morphogenetic plan. We further hypothesize that these effectors are conserved in zebrafish and that loss or gain of these signals can partially phenocopy the lower jaw defects observed in Hand2/Twist1 double conditional knockout mouse embryos. In both mouse and zebrafish, Hand2 is expressed in cranial neural crest cell (NCC)-derived mesenchyme within the distal mandibular pharyngeal arch, from which bone and cartilage of the lower jaw arise. We have shown that conditional deletion of the Hand2 gene within NCCs of mice results in patterning changes in mandibular arch-derived structures. In contrast to Hand2, the gene encoding the bHLH factor Twist1 is expressed in the NCC-derived mesenchyme of the proximal and intermediate mandibular arch. Targeted inactivation of Twist1 in NCCs leads to cell death in migrating NCCs and subsequent jaw defects. While Hand2 and Twist1 are thought to mark two distinct arch domains, our Preliminary Data illustrates that the expression domains of these two bHLH factors significantly overlap in the mandibular arch. Further, we show in Preliminary Data that deletion of both Hand2 and Twist1 within the overlapping domain almost completely abolishes organized lower jaw development. In this proposal, we will address the function of Hand2 and Twist1 in this overlapping domain in three Specific Aims. In Aim 1, we will, for the first time, define the function of Twist1 in post-migratory NCC patterning throughout the mandibular arch mesenchyme. Armed with this knowledge, Aim 2 will analyze the cellular and molecular changes that accompany loss of both Hand2 and Twist1 in their overlapping expression domain. We will also use RNA-seq to identify effectors of this combined signaling network. In Aim 3, we will determine the function of validated mediators using morpholino knockdown and overexpression in zebrafish. By coupling our cellular and molecular analysis of Hand2/Twist1 conditional knockout mice with our functional analysis of Hand2/Twist1 effectors in zebrafish, we will uncover novel regulatory mechanisms that establish an organizing region in the mandibular arch that ultimately directs lower jaw morphogenesis and whose disruption can lead to human facial birth defects. We expect these findings to shift the paradigm of how investigators view both mandibular arch patterning and the prospects of meaningful regenerative therapies.